RESUMEN
Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 plays a role in bacterial heavy metal resistance and catalyzes the reduction of arsenate to arsenite. The structures of the oxidized and reduced forms of ArsC were solved. ArsC has the PTPase I fold typical for low molecular weight tyrosine phosphatases (LMW PTPases). Remarkably, kinetic experiments show that pI258 ArsC also catalyzes the tyrosine phosphatase reaction in addition to arsenate reduction. These results provide evidence that ArsC from pI258 evolved from LMW PTPase by the grafting of a redox function onto a pre-existing catalytic site and that its evolutionary origin is different from those of arsenate reductases from Escherichia coli plasmid R773 and from Saccharomyces cerevisiae. The mechanism proposed here for the catalysis of arsenate reduction by pI258 ArsC involves a nucleophilic attack by Cys 10 on arsenate, the formation of a covalent intermediate and the transport of oxidative equivalents by a disulfide cascade. The reaction is associated with major structural changes in the ArsC.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bombas Iónicas , Complejos Multienzimáticos , Plásmidos , Proteínas Tirosina Fosfatasas/metabolismo , Staphylococcus aureus/enzimología , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Arseniato Reductasas , ATPasas Transportadoras de Arsenitos , Catálisis , Cristalografía por Rayos X , Datos de Secuencia Molecular , Oxidación-Reducción , Conformación Proteica , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Staphylococcus aureus/genéticaAsunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas/química , Bombas Iónicas , Complejos Multienzimáticos , Resonancia Magnética Nuclear Biomolecular , Staphylococcus aureus/enzimología , ATPasas Transportadoras de Arsenitos , Carbono/química , Hidrógeno/química , Nitrógeno/química , Oxidación-Reducción , Conformación Proteica , Proteínas Recombinantes de Fusión/químicaRESUMEN
Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Fabaceae/química , Lectinas/química , Lectinas/metabolismo , Plantas Medicinales , Secuencia de Aminoácidos , Sitios de Unión , Quitina/química , Quitina/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Disacáridos/química , Disacáridos/metabolismo , Evolución Molecular , Galactosa/metabolismo , Glicosilación , Enlace de Hidrógeno , Lactosa/metabolismo , Lectinas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Peptidilprolil Isomerasa de Interacción con NIMA , Oligosacáridos/química , Oligosacáridos/metabolismo , Isomerasa de Peptidilprolil , Lectinas de Plantas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Alineación de Secuencia , Especificidad por Sustrato , Trisacáridos/química , Trisacáridos/metabolismoRESUMEN
The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods. The thermodynamic stability was measured at different temperatures combining denaturant and thermally induced unfolding. It was found that both proteins denature in a two-state equilibrium (native dimer versus unfolded monomer) and that CcdA has a significantly lower thermodynamic stability. Using a numerical model, which was developed earlier by us, and on the basis of the determined thermodynamic parameters the concentration dependence of the denaturation transition temperature was obtained for both proteins. This concentration dependence may be of physiological significance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression is controlled by autoregulation. The influence of DNA on the thermal stability of the two proteins was probed. It was found that cognate DNA increases the melting temperature of CcdA. In the presence of non-specific DNA the thermal stability was not changed. The melting temperature of CcdB was not influenced by the applied double-stranded oligonucleotides, neither cognate nor unspecific.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Genes Bacterianos/genética , Plásmidos/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Rastreo Diferencial de Calorimetría , Dicroismo Circular , ADN/genética , ADN/metabolismo , ADN/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Escherichia coli/química , Escherichia coli/genética , Fluorescencia , Guanidina/farmacología , Concentración de Iones de Hidrógeno , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Oligodesoxirribonucleótidos/farmacología , Regiones Operadoras Genéticas/genética , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Especificidad por Sustrato , Temperatura , Termodinámica , Urea/farmacologíaRESUMEN
Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite). In order to study the structure of ArsC and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in Escherichia coli. In this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. A reversed-phase method was developed to separate and analyze the many different forms of ArsC. The oxidation state and the methionine oxidized forms were determined by mass spectroscopy.
Asunto(s)
Adenosina Trifosfatasas/aislamiento & purificación , Bombas Iónicas , Complejos Multienzimáticos , Staphylococcus aureus/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , ATPasas Transportadoras de Arsenitos , Cromatografía Liquida , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite), which is subsequently extruded from the cell. ArsC couples to thioredoxin, thioredoxin reductase, and NADPH to be enzymatically active. A novel purification method leads to high production levels of highly pure enzyme. A reverse phase method was introduced to systematically analyze and control the oxidation status of the enzyme. The essential cysteinyl residues and redox couple in arsenate reductase were identified by a combination of site-specific mutagenesis and endoprotease-digest mass spectroscopy analysis. The secondary structures, as determined with CD, of wild-type ArsC and its Cys mutants showed a relatively high helical content, independent of the redox status. Mutation of Cys 10, 82, and 89 led to redox-inactive enzymes. ArsC was oxidized in a single catalytic cycle and subsequently digested with endoproteinases ArgC, AspN, and GluC. From the peptide-mass profiles, cysteines 82 and 89 were identified as the redox couple of ArsC necessary to reduce arsenate to arsenite.
Asunto(s)
Adenosina Trifosfatasas/química , Bombas Iónicas , Complejos Multienzimáticos , Staphylococcus aureus/enzimología , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Secuencia de Aminoácidos , ATPasas Transportadoras de Arsenitos , Catálisis , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Dicroismo Circular , Cisteína/genética , Escherichia coli/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Estructura Secundaria de Proteína , Reductasa de Tiorredoxina-Disulfuro/biosíntesis , Reductasa de Tiorredoxina-Disulfuro/genéticaRESUMEN
The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB. Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent formation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Citotoxinas/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , ADN/metabolismo , Inhibidores de Topoisomerasa II , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Girasa de ADN , ADN-Topoisomerasas de Tipo II/genética , Hidrólisis , Mutación , Unión ProteicaRESUMEN
Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.
Asunto(s)
Ancylostoma/enzimología , Factores de Coagulación Sanguínea/antagonistas & inhibidores , Coagulación Sanguínea , Proteínas del Helminto/genética , Inhibidores de Serina Proteinasa/genética , Secuencia de Aminoácidos , Ancylostoma/genética , Animales , Sitios de Unión , Clonación Molecular , ADN Complementario/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Tromboplastina/metabolismoRESUMEN
We describe a novel phage display system that affords the surface expression and hence affinity selection of cDNAs. The strategy is based on a new approach to functionally display proteins on filamentous phage through the attachment to the C-terminus of the minor coat protein VI. The utility of the method was evaluated using a cDNA library derived from the parasite Ancylostoma caninum. cDNA sequences were fused in each of the three reading frames to the 3'-end of the M13 gene VI expressed by a phagemid vector. Phages rescued from this cDNA expression library were subjected to biopanning against two serine proteases, trypsin and the human coagulation factor Xa. This led to the identification of cDNAs encoding novel members of two different families of serine protease inhibitors. The authenticity of the cDNA selected with trypsin as the target was demonstrated by purifying the encoded potent Kunitz-type inhibitor from an Ancylostoma caninum extract. The rapid isolation of specific cDNAs with the protein VI monovalent display system should facilitate the search for novel biologically important ligands.
Asunto(s)
Cápside/genética , ADN Complementario/genética , Secuencia de Aminoácidos , Ancylostoma/enzimología , Ancylostoma/genética , Animales , Secuencia de Bases , Biotinilación , Western Blotting , Factor Xa/metabolismo , Biblioteca de Genes , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Serina Endopeptidasas/genética , Inhibidores de Serina Proteinasa/genética , Tripsina/metabolismo , Inhibidores de Tripsina , alfa-Amilasas/antagonistas & inhibidoresRESUMEN
Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.
Asunto(s)
Inhibidores del Factor Xa , Péptidos/genética , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Secuencia de Bases , Isótopos de Carbono , Clonación Molecular , Fermentación , Vectores Genéticos , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Biosíntesis de Péptidos , Pichia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Selección Genética , GarrapatasRESUMEN
Glial fibrillary acidic protein (GFA) expression was induced in rat C6 glioma in chemically defined medium by the addition of N6, O2'-dibutyryl cyclic AMP (dbcAMP). Induction was dependent on the increase in intracellular cyclic AMP (cAMP), which was linearly correlated with added dbcAMP. Contrary to GFA mRNA synthesis, which can be obtained by cAMP-dependent and -independent pathways, translation of mRNA into GFA was observed only above a cellular cAMP concentration of approximately 0.2 fmol/cell. dbcAMP stimulation did not affect the vimentin concentration, which remained at a low level, but changed the cellular morphology from a bipolar to a stellate shape. A similar morphological change was observed after stimulation of C6 with lipopolysaccharide (LPS). However, LPS did not significantly increase the intracellular concentration of cAMP and the LPS-induced mRNA was not translated into GFA. Our results indicate that GFA synthesis is regulated at the mRNA level and at the translational level and that a cAMP-dependent mechanism determines the ultimate synthesis of GFA by a yet unknown mechanism.